OPK-2,007-0;Y 

BruggemannChemical U.S., hc. 

Subsidiary of the L. Bruggemann KG Germany 

15 Reese Avenue - Suite 200 

Newtown Square, PA 19073 " USA 

March 7, 2011 

Via Overnight Delivery 

Bruggemann 

Chemical 

u~0 mi~u~0 p~ius0 n~2 4 ~ 

Document Control Office (DCO) (7407M) 

Office of Pollution Control and Toxics (OPPT) 

U.S. Environmental Protection Agency (EPA) 

EPA East Building, Room E6428 (202-564-8930) 

1201 Constitution Ave., NW 

Washington, D.C. 20004-3302 

Attn: TSCA Section 4 

40 C.F .R. 799.5087(Docket # EPA-HQ-OPPT-2007-0531) 

~.3 

.cRe: Letter of Intent/Submission of Data in Response to TSCA Section 4 Test Rule; 

Methanesulfinic acid, hydroxyl-, monosodium salt (CASRN 149-44-0) 

Dear Sir or Madam: 

By this letter, BruggemannChemical U.S., Inc. (BruggemannChemical)1 is notifying the 

U.S. Environmental Protection Agency (EPA) of its intent to satisfy the testing 

requirements for Methanesulfinic acid, hydroxyl-, monosodium salt with CASRN 149-

44-0 (also known as "sodium hydroxymethanesulfinate" or "sodium formaldehyde 

sulfoxylate") specified in the Toxic Substances Control Act (TSCA) section 4 test rule 

that appears in 40 C.F.R. § 799.5087 (Test Rule). According to the Test Rule, a 

"Bacterial Reverse Mutation Test (in vitro) : 40 CFR 799.9510" must be conducted for 

the chemical substance. 

As documented in the enclosed files, we have recently conducted the prescribed testing 

for the substance in accordance with OECD Guideline 471 which is consistent with the 

referenced EPA method. As noted in the study report, the test item is considered to be 

non-mutagenic in the bacterial reverse mutation test. Accordingly, we trust that through 

the provision of the enclosed study report and robust summary BruggemannChemical has 

fulfilled its obligations under the Test Rule. 

1 BruggemannChemical U.S., Inc. is located at 15 Reese Avenue, Suite 200, Newtown Square, PA 19073, 

telephone: (610) 353-6840 . BriiggemannChemical U.S ., Inc. is the U.S . subsidiary of 

BrtiggemannChemical which is headquartered in Germany. 

CONTAINS NO CEjt 

a

_ 

Edward 1. McDade, III For Deposit of LISD: 

Vice President Wachovia Bank 

Account: 2100012829009 

ABA: 031201467 

0 9001/14001 Swift Code: TNBTUS33 

REG . NR . 1499-02 

For Deposit of EUR 

Baden-Wurttembergische Bank in Heilbronn 

(BLZ 60050101) i .e . 

Account #1430019 

ABA: DE066 050 101 00014 300 19 

Swift Code: SOLADE ST 

Will 

Phone 610 353 9852 

Fax 610 353 9853 

info aClbrueggeman.com 

www.brueggemann .com 
Should you have any questions regarding this matter, or desire further information, please 

do not hesitate to contact Mr. Ed McDade at (610) 353-9852 or 

Ed.McDade@brueggemann.com. 

Sincerely, 

Mr. David E. Wilson 

President 

Enclosures 

1. Final Report - Determination of the mutagenic potential of NHS (Sodium 

hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following 

OECD 471 and EU B 13/14. 

2. IUCLID Endpoint Study Record: Genetic toxicity in vitro. 001 . 
,*nr~ ,  

~ r~eE, jr 

Final Report 

Determination of the mutagenic potential of NHS (Sodium 

hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 

IT 4 4 

07120702G803 
Final Report of LAUS GmbH No. : AB07120702G803 

Auf der Schafweide 20, D-87489 Kirrweiler 

Tel. ++49-8321-96299-0, Fax 98299-29, e-mail: infoClaus .de Date: 02. May 2008 

Final Report 

ORIGINAL . Z OF _4Z 

Title : Determination of the mutagenic potential of NHS (Sodium 

hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13114 

Project No. : 07120702G803 

Sponsor: BrUggemann Chemical 

UM 

Salzstraf3e 131 

D-74076 Heilbronn 

Monitor: Dr. Franz NGRlein 

Test Item : NHS (Sodium hydroxymethanesull"inate) 

Batch No. : 07041205 

Test Facility: LAUS GmbH 

Auf der Schafweide 20 

D-67489 Kirrweiler 

Study Director: Dipl. Chem . Jbrg Paufus 

Deputy Study Director: Dipl. Ing . Anette Rudolf 

Time Table: Study Plan dated: 

Experimental Starting Date: 

Experimental Completion Date: 

Draft Report: 

Final Report: 

08. Jan. 2008 

16. Jan. 2008 

25. Jan. 2008 

29. Jan. 2008 

02 . May 2008 
Final Report of LAUS GmbH 

Auf der Schafweide 20, D-67489 Kircrweiler 

No. : AB07120702G803 

Tel . ++49-8321-98299-0, Fax 96299-29, e-mail: info®Iaus .de f Date: 02. May 2008 

This page was intentionally left blank for statement of sponsor or submitter. 

page 2 of 37 
Final Report of LAUS GmbH 

Ad der Schalwaide 20, D-67489 Kirrweiler 

No. : AB07120702G803 

Tel . ++49-6321-96299-0, Fax 96299-29. e-mail : infoQlaus.de I Date: 02 . May 2008 

I GLP-COMPLIANCE CLAIM 

It is hereby declared that all tests were made in accordance with the Revised OECD Principles of Good Laboratory Practice" (Paris, 1997) as stated in the following guidelines: 

OECD Principles of Good Laboratory Practice, adopted by Council on 26th November 1997; Environment Directorate, Organisation for Economic Cooperation and Development, Paris 1998 

Directive 2004/10/EC of the European Parliament and of the Council of 11 February 

2004 on the harmonisation of laws, regulations and administrative provisions relating 

to the application of the principles of good laboratory practice and the verification of 

their applications for tests on chemical substances (codified version) 

ChemikaliengesetZ (Chemicals Act) of the Federal Republic of Germany (ChemG) 

§19 and annexes 1 and 2 in the version of 25. July 1994 (BGBI. I p. 1703), amendments as of 08. May 2001 (BGBI. I, p. 843) and 20. Jun. 2002 (BGBI. I pp . 2090- 2130) . 

There were no circumstances that may have affected the quality or integrity of the study. 

This report contains data which were not determined under GLP conditions : 

Cytotoxicity (see annex 4) of the test item was determined before initiation of the study. 

Dipl. ~h,6m ."Jdi`g 

Study 

G ~ 1 . ; ;,Y 2008 

Date 

Personnel involved in the performance of the study: 

Isabell Andres Technical Assistant Biology 

Anette Rudolf Dipl.-Ing . Environmental Protection 

JUrgen Schlinck Technical Assistant Biology 

page 3 of 37 
Final Report of LAUS GmbH 

Auf der schalWeide 20, 0-67489 Kirrweifer 

No. : AB07120702G803 

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2 QUAL1Ty ASSURANCE STATEMENT 

Title : Determination of the mutagenic potential of NHS (Sodium 

hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 

Test Item: NHS (Sodium hydroxymethanesulfinate) 

Batch No. : 07041205 

Project No. : 07120702G803 

This study has been inspected by the quality assurance unit according to the principles of 

Good Laboratory Practice . Study Plan and Final Report were checked at the dates given 

below, the Study Director and the management were informed with the corresponding report. 

Also, the performance of the study was inspected, and findings were reported to Study 

Director and management. The inspection of short-term studies (duration less than one week) is carried out as audit of process concerning major technical phases of at least one 

similar test. Frequency is once or more a quarter. 

The study was conducted and the reports were written in accordance with the Study Plan 

and the Standard Operating Procedures of the test facility . 

Deviations from the Study Plan were acknowledged and assessed by the Study Director 

and included in the Final Report. 

The reported results reflect the raw data of the study. 

Verified procedure Date of Inspection Audit report no. Audit report dated 

Study plan 09. Jan . 2008 080109-01 09 . Jan . 2008 

Performance of study 23. Jan. 2008 080123-02 23 . Jan . 2008 

Draft report 07. Feb. 2008 080207-01 07 . Feb. 2008 

Final report 01 . May 2008 080501-03 01 . May 2008 

~' % , .r ; ' 2008 

Jutta Paulus Date 

Quality Assurance 

page 4 of 37 
Final Report of LAUS GmbH No. : AB07120702G803 

Auf der Schafweide 20, D-67489 Kinweiler 

Tel . ++49-6321-96299-0, Fax 96299-29, e-mail: info@laus .de Date: 02. May 2008 

Table of Contents 

1 GLP-COMPLIANCE CLAIM 3 

2 QUALITY ASSURANCE STATEMENT 4 

3 SUMMARY g 

4 PURPOSE OF THE STUDY 10 

5 LITERATURE 10 

6 MATERIALS AND METHODS 10 

6 .1 Test Item 10 

6 .1 .1 Specification 10 

6 .1 .2 Storage 11 

6 .1 .3 Preparation 11 

6 .2 Reference Items 11 

6 .2 .1 NPD 11 

6 .2 .2 Sodium azide 11 

6 .2 .3 BaP 11 

6 .2 .4 2-AA 11 

6.3 Test System 11 

6 .3 .1 Specification 11 

6 .3 .2 Origin and Culture 11 

6 .4 Chemicals 12 

6 .4,1 Nutrient broth for overnight culture 12 

6,4 .2 Isotonic sodium chloride solution for dilution purposes 12 

6 .4 .3 Vogel-Bonner-Medium 20fold 12 

6 .4 .4 Glucose solution 40% 12 

6 .4 .5 Minimal Glucose Agar 12 

6 .4 .6 Biotin Agar 12 

6 .4 .7 Histidine-Biotin-Agar 12 

6.4 .8 Ampicillin-Agar 12 

6.4 .9 Ampicillin-tetracycline plates 12 

6.4 .10 Nutrient agar plates 13 

6.4 .11 Basis for Top-Agar and Maximal-Soft-Agar 13 

6.4 .12 Histidine-Biotin-solution 0 .510.5 millim. (use: top agar) 13 

6.4 .13 Histidine-Biotin-solution 5 millim .l 0.5 millim . (use: maximal-soft-agar) 13 

6.4 .14 Phosphate buffer 13 

6.4 .15 Salt solution for S9-Mix 13 

6 .4 .16 NADP-solution for S9-Mix 13 

6 .4 .17 Glucose-6-phosphate solution for S9-Mix 13 

6.4 .18 S9-Mix 13 6.4 .19 S9 13 

6.5 Test Vessels 15 

page 5 of 37 
Final Report of LAUS GmbH 

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6.6 Instruments and Devices 

7 PERFORMANCE OF THE STUDY 

7.1 Culture of Bacteria 

7.2 Description of the Method 

7 .2 .1 Preparations 

7 .2 .2 Experimental Parameters 

7 .2 .3 Description of the Method 

7.3 References 

7 .3 .1 Genotype Confirmation 

7 .3 .2 Determination of Titre 

7 .3 .3 Toxicity Control 

7 .3 .4 Sterility Control 

7 .3 .5 Positive Controls 

7.4 Evaluation 

8 FINDINGS 

8.1 First Experiment 

8 .1 .1 Confirmation of the criteria 

8 .1 .2 Mutagenicity 

8 .1 .3 Survey of the Findings 

8.2 Second Experiment 

8,2 .1 Confirmation of the Criteria 

8.2 .2 Solubility and Toxicity 

8.2 .3 Mutagenicity 

8 .2 .4 Survey of the findings 

9 RESULTS 

10 DISCUSSION 

11 DEVIATIONS 

11 .1 Deviations from the Study Plan 

11 .2 Deviations from the Guideline 

12 RECORDING 

13 ANNEX 1 : COPY OF GLP CERTIFICATE 

14 ANNEX 2: DATA OF THE FIRST EXPERIMENT 

14.1 Determination of Titre 

14.2 Sterility Control 

14.3 Spontaneous Revertants 

14 .3 .1 Water 

14 .3 .2 DMSO 

14.4 Positive Controls 

laus .de 

No. : AB07120702G803 

Date: 02. May 2008 

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page 6 of 37 
14.6 Test Item NHS (Sodium hydroxymethanesuiffnate) 

14 .5 .1 Mutagenicity Test 

15 ANNEX 3: DATA OF THE SECOND EXPERIMENT 

15.1 Determination of Titre 

15.2 Sterility Control 

15.3 Spontaneous Revertants 

15 .3 .1 Water 

15 .3 .2 DMSO 

15.4 Positive Controls 

15.5 Test Item NHS (Sodium hydroxymethanesulflnate) 

15 .5 .1 Mutagenicity Test 

16 ANNEX 4: DATA OF THE PRE-TEST (CYTOTOXICITY) 

16.1 Experimental Parameters 

16.2 Determination of Titre 

16.3 Toxicity Control 

16.4 Assessment 

17 ANNEX 5: COMPARISON WITH HISTORICAL DATA 

18 ANNEX 6: GENOTYPE CONFIRMATION 

18.1 Histidine Requirement 

18.2 Ampicillin-(Tetracyclinee)-Resist (pKM 101, pAQ1) 

18.3 UV-Sensitivity (uvrB) 

18.4 Crystal Violet Sensitivity (deep roughlrfa) 

28 

28 

30 

30 

30 

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31 

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32 

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3 SUMMARY 

Final Report of LAUS GmbH 

Auf der Schafweide 20, D-B7489 Kirrweiler 

No. : AB07120702G803 

Tel . ++49-6321-96299-0, Fax 96299-29, e-mail : info@laus .de I Date: 02. May 2008 

Study: Determination of the mutagenic potential of NHS (Sodium 

hydroxymethanesulfinate) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 

Sponsor: Brtiggemann Chemical 

UM 

Salzstraf3e 131 

D-74076 Heiibronn 

Monitor: Dr. Franz Nuf3lein 

Test Facility: LAUS GmbH 

Auf der Schafweide 20 

D-67489 Kirrweiler 

Project No. : 07120702G803 

Study Plan No. : PP07120702G803 

Findings: 

Two valid experiments were performed. 

First Experiment: 

Five concentrations of the test item, dissolved in deionised water (ranging from 5047 to 

51 pg/plate) were used . Five genetically manipulated strains of Salmonella fyphimurium 

(TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the 

presence and in the absence of a metabolic activation system (S9) for 48 hours, using the 

plate incorporation method . 

None of the concentrations caused an increase in the number of revertant colonies in the 

tested strains . The test item didn't show any mutagenic effects in the first experiment. 

No signs of toxicity towards the bacteria could be observed. 

The sterility control and the determination of the titre didn't show any inconsistencies . The 

determined values for the spontaneous revertants of the negative controls were in the 

normal range. All positive controls showed mutagenic effects with and without metabolic 

activation. 

page 8 of 37 
Second Experiment: 

To verify the results of the first experiment, a second experiment was performed, using 

three concentrations of the test item (ranging from 5031 to 1258 irg/plate) and a modification in study performance (pre-incubation method). 

The test item didn't show mutagenic effects in the second experiment, either. 

No signs of toxicity towards the bacteria could be observed. 

The sterility control and the determination of the titre didn't show any inconsistencies . The 

determined values for the spontaneous revertants of the negative controls were in the 

normal range. All positive controls showed mutagenic effects with and without metabolic 

activation . 

Under the conditions of the test, the test item didn't show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 . 

Therefore, no concentration-effect relationship could be determined .' 

The test item NMS (Sodium hydroxymethanesulfinate) is considered as 

"not mutagenic under the conditions of the test". 

page 9 of 37 
Final Report of LAUS GmbH 

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4 PURPOSE OF THE STUDY 

This study was performed in order to evaluate the mutagenic potential of NHS (Sodium 

hydroxymethanesulfinate) . The test was chosen on behalf of the sponsor for registration 

purposes following Chemicals Law. 

5 LITERATURE 

The study was conducted in accordance with the following guidelines: 

OECD Guidelines for the Testing of Chemicals 

Bacterial Reverse Mutation Test" 

Part 471, adopted July 218t, 1997 

EU-Guideline 671548 EWG, last changed by 29th amendment, Annex V, Method 

B .13/14 "Mutagenicity - Salmonella typhimurium, reverse mutation 

assay", adopted May 19th, 2000 

Corresponding SOP of LAUS GmbH 

SOP 118 008 03 edition 6, dated 13. Nov. 2006 

Bestimmung des erbgutverandernden Potentials mit dem Bacterial-ReverseMutation-Test (AMES-Test)" 

For historic data, the following literature was included: 

Revised Methods for the Salmonella mutagenicity test" by Dorothy Maron and 

Bruce Ames, Mutation Research 113 (1983), S. 173 - 215 

6 MATERIALS AND METHODS 

6 .1 Test Item 

Prof. 

6 .1 .1 Specification 

The following information concerning identity and composition of the test item were 

vided by the sponsor. 

Name 

Batch no 

Appearance 

Composition 

CAS-No. 

EINECS-No. 

Molecular formula 

Molecular weight 

Purity 

NHS (Sodium hydroxymethanesulfinate) 

07041205 

white powder 

main component Sodium hydroxymethanesulfinate 

4 .4 - 6 % Na-hydroxymethansulfonat 

1 .9 - 5% Na-sulfit 

2 .4 - 3% Na-sulfat 

1 .2 - 2 % Na-carbonat 

11 .5 -15 .5 % (crystal) water 

149-44-0 

2057394 

CH303S"Na resp . CH303S*Na x 2 H20 

154.1 glmol 

99.3% (dihydrate), 76 .1% (anhydrous) 

propage 10 of 37 
Homogeneity 

Volatility 

Stability 

Solubility 

Date of+production 

Date of expiry 

Storage conditions 

Hazard information 

R-phrases 

S-phrases 

Final Report of LAUS GmbH 

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homogeneous 

not volatile 

H20 96h, EtOH, acetone, CH3CN, DMSO not stated 

H20 >1gIL EtOH, acetone, CH3CN, DMSO not stated 

12. Apr. 2007 

12. Apr. 2009 

room temperature 20 t 5 °C 

none 

none 

none 

6 .1 .2 Storage 

The test item was stored in a closed vessel at room temperature . 

6 .1,3 Preparation 

Preparation was the same for both experiments . 

On the day of the start of the experiment, a stock solution containing 50 gIL of the test 

item in deionised water was prepared . Water was chosen as solvent, because the test 

item was completely soluble, and this solvent doesn't have any effects on the viability of 

the bacteria or the number of spontaneous revertants. The stock solution was used to 

prepare the geometric series of the concentrations to be tested . Each solution was membrane 5ltrated to accomplish sterility . 

6.2 Reference Items 

The following mutagenic substances were used as positive controls: 

6.2.1 NPD 

4-Nitro-1,2-phenylene diamine, C6H7N302; CAS-No. : 99-56-9 

6 .2 .2 Sodium azide 

Sodium azide, NaN3; CAS-Nr. : 26628-22-8 

6 .2 .3 BaP 

Benzo-a-pyrene, C2oH12 ; CAS-Nr. : 50-32-8 

6.2 .4 2-AA 

2-Amino-anthracene, C14H11N; CAS-Nr. : 613-13-8 

6.3 Test System 

6.3 .1 Specification 

Species Salmonella typhimurium LT2 

Strains TA 1535, TA 97a, TA 98, TA 100 and TA 102 

Mutations TA97a: hisD6610, TA 98: hisD3052 TA 100 und TA 1535 hisG46, TA102: 

hisG428, TA97, TA98 and TA100 contain uvr8; TA97, TA98, TA100 and 

TA102 contain pKM 101 and rfa. 

6 .3 .2 Origin and Culture 

Salmonella typhimurium (all strains used) were obtained from Prof. Ames (date of arrival: 

Feb. 25th 2002 (TA97a, 98, 102), June 23`d 2001 (TA 100) and Jan. 29°h, 2001 (TA1535)) 

and stored as stock cultures at - 80°C. One day before the start of each experiment, one 

vial per strain to be used was thawed and put into a culture vessel containing nutrient 

page 11 of 37 
Final Report of LAUS GmbH 

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broth. After incubation over night at 37 C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the 

titre . 

6.4 Chemicals 

The purity of the chemicals which were used were either analytical grade" or for microbiological purposes" . Every substance was controlled upon arrival in the test facility . 

All solutions and media were sterilized, either by autoclaving (121 ~C, 20 minutes) or by 

membrane filtration . 

The weights may differ from the theoretical value (difference < 5 %) . Additionally, differing 

specifications (e.g . water of crystallization) may be used. 

6.4 .1 Nutrient broth for overnight culture 

Nutrient broth Merck 5443 2.8 g 

H20 demin . ad 350 mL 

6.4.2 Isotonic sodium chloride solution for dilution purposes 

Sodium chloride 0.9 g 

HZO demin . ad 100 mL 

6 .4 .3 Vogel-Bonner-Medium 20fold 

Magnesium sulphate (MgS04''7H20) 4 .0 g 

Citric acid mono hydrate 40 .0 g 

Potassium phosphate, dibasic (anhydrous) (K2HP04) 200 .0 g 

Sodium ammonium phosphate, monobasic, tetra hydrate (Na(NH4)HP04*4H20) 70 .0 g 

H20 demin. ad 1000.0 mL 

6.4 .4 Glucose solution 40% 

Glucose 400.0 g 

H20 demin. ad 1000.0 mL 

6.4 .5 Minimal Glucose Agar 

Vogel-Bonner-Solution 20fold 500 .0 mL 

Glucose solution 40% 500.0 mL 

H20 demin . 9000 .0 mL 

Agar 150.0 g 

6.4.6 Biotin Agar . 

Minimal-Glucose-Agar, 80 C 500 .0 mL 

Biotin solution 0.5 millimolar 3 .0 mL 

6.4.7 Histidine ;Biotin-Agar 

Biotin-Agar, 80 C 350.0 mL 

Histidine solution 0.5% 3.5 mL 

6 .4 .8 Ampicillin-Agar 

Histidine-biotin agar, 80 °C 250.0 mL 

Ampicillin solution 0.8% 0.6 mL 

6.4 .9 Ampicillin-tetracycline plates 

Ampicillin agar, 80 C 50 .0 mL 

Tetracycline solution 0 .8% 0.01 mL 

page 12 of 37 
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6.4 .10 Nutrient agar plates 

Nutrient broth Merck 5443 0.8 g 

Sodium chloride (NaCl) 0.5 g Agar 1 .52 g 

HZO demin . 100 mL 

6 .4 .11 Basis for Top-Agar and Maximal-Soft-Agar 

Agar 6 g 

Sodium chloride (NaCl) 5 g 

H20 demin . ad 1000 mL 

6.4.12 Histidine-Biotin-solution 0.5/0 .5 millim . (use: top agar) 

D-Biotin (MG 247.3) 12 .36 m 9 L-Histidine* HCI"`1 H20 (MG 209.7) 10.5 mg 

H20 demin ., 90°C ad 100.0 mL 

To 100 mL basis (see 7.4 .11), 10 mL histidine-biotin-solution 0.5/0 .5 millim . are added. 

6.4.13 Histidine-Biotin-solution 5 millim ./ 0 .5 millim . (use: maximal-soft-agar) 

D-Biotin (MG 247.3) 12 .36 mg 

L-Histidine"' HCI*1 H20 (MG 209.7) 105 mg 

H20 demin., 90°C ad 100.0 mL 

To 100 mL basis (see 7.4 .11),10 mL histidine-biotin-solution 5/0 .5 millim . are added . 

6.4 .14 Phosphate buffer 

Sodium di-hydrogen phosphate monohydrate NaH2P04*H20 0.3312 g 

Di-sodium hydrogen phosphate NaZHP04 2 .5 g 

H20 demin . ad 100 .0 mL 

The pH of the solution is adjusted to 7 .4 t 0.1 . 

6.4 .15 Salt solution for S9-Mix 

Potassium chloride (KCI) 1 .23 g 

Magnesium chloride hexahydrate MgCl2'`6H20 0 .814 g 

H20 demin. ad 10.0 mL 

6.4 .16 NADP-solution for S9-Mix 

NADP (MR = 765.4 g/mol) 766 mg 

H20 demin. 10 mL 

6.4 .17 Glucose-6-phosphate solution for S9-Mix 

Glucose-6-phosphate (MR = 282 g/mol) 564 mg 

H20 demin., sterile ad 2 mL 

6.4 .18 S9-Mix 

Phosphate buffer 22,5 mL 

0.1m NADP-solution 1 .0 mL 1 m-G6P-so[ution 0.125 mL Salt solution 0.5 mL Rat liver S9, 4°!0 1 .0 mL 

6.4 .19S9 

S9 was obtained by Trinova Biochem, Gief3en . 

Batch no : 2118, 2081, 2113 

page 13 of 37 
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Specification : produced from the livers of male Sprague-Dawley rats which 

were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally . 

page 14 of 37 
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No. : AB07120702G803 

Date: 02 . May 2008 

6.5 Test Vessels 

All vessels used are made of glass or sterilizable plastic. They were sterilised before use 

by heating to 180 °C (two hours) or autoclaving. 

The following vessels were used : 

Schott-bottles, glass vials, and culture flasks for solutions and media 

Plastic petri plates 

Glass tubes (with Kapsenberg caps) for TopAgar-bacteria-substance mix 

6.6 Instruments and Devices 

The following instruments and devices were used in the performance of the study. 

Autoclave MLS 3020 

Magnetic stirrer and magnetic stir bars 

Scales Mettler PB 5001-S02 

Analytical scales MT AB 184 SA 

Incubation chamber Heraeus, adjustable to 37 °C 

Table water bath GFL, adjustable to 45 °C 

Heating chamber Memmert 1 

Orbital shaker GFL 3005 

Adjustable pipettes with sterile tips, LAUS No. 8 (0,2-2ml), LAUS No. 22 (20-200p1), 

LAUS No. 32-33 (500N1), LAUS No. 34-38 (100N1) 

Glass thermometer LAUS No. 20050117-74; 20020912-22 ; 20020912-15 

Glass measuring cylinders, 100 and 1000 mL 

Glass measuring flasks, 25, 500 and 1000 mL 

Membrane filters, 0.2 pm pore diameter 

Drigalski spatula 

Usage and, if applicable, calibration following the corresponding SOP in the current edition. 

page 15 of 37 
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7 PERFORMANCE OF THE STUDY 

7.1 Culture of Bacteria 

No. : AB07120702G803 

Date: 02. May 2008 

12 hours before the start of each experiment, a stock culture of each strain was thawed 

and an aliquot was placed into a culture flask containing 70 mL nutrient broth. The flasks 

were incubated at 37°C for 12 hours . 

7.2 Description of the Method 

7 .2 .1 Preparations 

In the days before each test, the media and solutions were prepared. Two days before the 

test, the plates were sterilised and the first batches poured. On the day before the test, the 

remaining plates were poured. 

On the day of the test, the overnight cultures were checked for growth. The incubation 

chamber was heated to 37 °C. The water bath and the heating block were turned to 43 °C . The table surface was disinfected . 

The S9 mix was freshly prepared and 

7 .2 .2 Experimental Parameters 

7.2.2 .1 First Experiment 

Date of treatment 

Concentrations tested 

Tester strains 

Incubation time 

Incubation temperature 

Tester strains 

Method 

References 

7.2 .2 .2 Second Experiment 

Date of treatment 

Concentrations tested 

Tester strains 

Incubation time 

Incubation temperature 

Method 

References 

stored at 0 °C. 

January 161h, 2008 

5047 1 1514/5051151 /51 Ng/plate 

TA97a, TA98, TA100, TA102, TA 1535 

48 hours 

37 °C 

TA97a, TA98, TA100, TA102, TA1535 

plate incorporation method 

see under 7.3.5 

January 23`4, 2008 

5031 I 2516 I 1258 pg/plate 

TA97a, TA98, TA100, TA102, TA1535 

48 hours 

370C 

pre-incubation method 

see under 7.3.5 

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7 .2 .3 Description of the Method 

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No. : AB07120702G803 

Date : 02. May 2008 

7 .2 .3.1 Plate incorporation method 

Per strain and dose, four plates with and four plates without S9 mix were used . 10 mL of 

the test solution of the appropriate concentration were membrane filtrated into sterile vessels . Top agar basis was melted in a microwave oven, after melting, 10 mL of histidinebiotin-solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the 

water bath at 45 °C . 

0 .1 mL of the appropriate solution of the test item were given into a sterile tube . After mix- ing with 0 .1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only for treatments without S9) or 0 .5 mL S9 mix, 2 mL Top-Agar were added . The mixture was gently vortexed, then poured on a minimal glucose plate and distributed evenly, 

using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C. 

7.2.3.2 Pre-incubation method 

Per strain and dose, four plates with and four plates without S9 mix were used . 10 mL of 

the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine- biotin-solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45 °C . 

0.1 mL of the appropriate solution of the test item were given into a sterile tube. After mix- ing with 0.1 mL overnight culture of the respective strain, 0 .5 mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an 

incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated 

through careful shaking . Then 2 mL top agar were added. The mixture was vortexed gen- tly, then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula . The plates were closed, covered with brown paper and left to harden for a few min- utes, then inverted and placed in the dark incubator at 37 °C . 

page 17 of 37 
7.3 References 

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7.3 .1 Genotype Confirmation 

Genotype confirmation is performed once a term. 

7.3 .1 .1 Histidine requirement 

Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilised wire loop . 

7.3 .1 .2 Ampicillin-Resistance (pKM 101) resp. ampicillin-tetracycline-resistance (pAQ1) 

The strains were streaked on ampicillin agar, TA102 on ampicillin-tetracycline agar. 

TA1535 was taking the function of control strain, since it is not ampicillin resistant . 

7.3 .1 .3 UV-sensitivity (uvr8) 

Two plates were streaked with the five strains, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light . The plates were 

irradiated for 8 seconds with a germicidal lamp (254 nm, 30W), keeping a distance of 

33 cm. Incubation over night at 37 °C followed . 

7 .3 .1 .4 Crystal violet sensitivity (deep rough) 

For each strain, two plates were used . 0.1 mL of bacteria suspension were mixed with 

2 mL Top-Agar and poured on nutrient agar. Sterile paper discs (Q1 9 mm), each soaked 

with 10 NI of crystal violet solution (0.1%) were placed into the middle of each plate, fol- lowed by incubation over night. 

7 .3 .1 .5 Spontaneous revertants 

Four replicates, with/without S9, for each solvent which was used in the test. 

7 .3 .2 Determination of Titre 

The titre was determined by dilution of the overnight culture using sodium chloride solution 

and placing 0 .1 mL on maximal-soft agar. It should give a density of 109 ceIIsImL (at the least) . 

7.3.3 Toxicity Control 

Performed analogously to the titre control with the maximum dose of test item with and 

without S9 on maximal-soft agar. 

7.3.4 Sterility Control 

Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar. 

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7.3 .5 Positive Controls 

Using diagnostic mutagens, four replicates were prepared . The stock solutions of the sub- stances were diluted to effect an application volume of 0.1 mUplate. 

7.3 .5 .1 Without metabolic activation system 

+ 4-Nitro-1,2-pheny(ene diamine 

Concentration per plate: 80 Ng ; strains TA 97a, TA 98 and TA 102 . 

+ Sodium azide 

Concentration per plate: 6 Ng; strains TA 100 and TA 1535 . 

7 .3 .5.2 With metabolic activation system 

+ Benzo(a)pyrene 

Concentration per plate: 40 Ng; strain TA 98. 

2-Aminoanthracene (2-AA) 

Concentration per plate: 3 pg ; strains TA 97a, TA 100, TA 102 and TA 1535. 

7.4 Evaluation 

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excelo) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction . 

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor >_ 2) in at least one strain can be observed . A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity . 

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8 FINDINGS 

The detailed data are listed in the annex. 

8.1 First Experiment 

No. : AB07120702G803 

Date: 02 . May 2008 

8 .1 .1 Confirmation of the criteria 

The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn't show any inconsistencies . The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see page 36). All positive controls (diagnostic mutagenes) 

showed mutagenic effects with and without metabolic activation . 

The test item was dissolved in deionised H20, preparing a stock solution containing 50 gIL (nom .) . No signs of toxicity towards the tested strains could be observed : the background 

lawn was visible, the number of revertant colonies was not significantly reduced . 

8 .1 .2 Mutagenicity 

No significant increase of the number of revertant colonies in the treatments with and with- out metabolic activation could be observed . No concentration-related increase over the 

tested range was found . 

Therefore the test item is stated as not mutagenic under the test conditions. 

To verify this result, a second experiment was performed using the pre-incubation method. 

page 20 of 37 
8 .1 .3 Survey of the Findings 

The mean revertant values of the four replicates are presented in the following table. 

Table 8.1-a Mean Revertants First Experiment 

Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

H 0 Mean 131 96 10 9 190 161 171 180 10 12 2 

sd 49 4 5 4 33 26 36' 72 1 3 

DMSO Mean 116 111 12 9 180 182 178 172 14 21 

sd 42 40 3 1 28 20 10~ 20 3 10 

Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Pos . Contr. sd 0 0 0 0 0 0 0 0 0 0 

f(l) 8.63 9.02 83.42 111 .2 5.27 5.50 5.62 5.82 100.1 47 .67 

Mean 119 110 12 7 164 140 82 157 10 16 

5047 pg/pl . sd 47 16 5 2 38 14 24 33 4 3 

f(l) 0.91 1 .15 1 .20 0.78 0 .86 0.87 0.48 0.87 1 .00 1 .33 

Mean 105 137 11 7 182 163 152 136 10 8 

1514 pg/pl . sd 23 23 2 5 39 46 46 57 1 1 

f(l) 0.80 1 .43 1 .10 0.78 0 .96 1 .01 0.89 0.76 1 .00 0.67 

Mean 136 122 10 8 168 143 125 117 9 10 

505 pg/pl. sd 32 41 3 2 

__ 

28 24 14 20 1 1 

f(l) 1 .04 1 .27 1 .00 0.89 0.88 0.89 0.73 0.65 0.90 0 .83 

Mean 98 123 10 10 134 176 130 132 11 11 

151 pg/pl . sd 40 20 2 

_ 

2 43 36 18 18 5 4 

f(l) 0.75 1 .28 1 .00 1 .11 0.71 1 .09 0.76 0.73 1 .10 0 .92 

Mean 119 139 12 6 193 167 170 149 14 9 

511aglpl . sd 30 22 5 

_ 

1 31 27 32 31 4 1 

f( l) l 0.91 ~ 1 .45 1 1 .20 0.67 1 .02 ~ 1 .04 0. 99 0. 83 1 .40 0. 75 

In this table, "1001" represents "> 1000" . 

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8.2 Second Experiment 

laus .de 

No. : AB07120702G803 

Date: 02. May 2008 

8 .2 .1 Confirmation of the Criteria 

The treatments for the confirmation of the genotype, the sterility control and the determination of the titre didn't show any inconsistencies . The determined values for the spontaneous revertants of the negative controls were in the normal range -of the test laboratory. 

All positive controls showed mutagenic effects with and without metabolic activation . 

8 .2 .2 Solubility and Toxicity 

The test item was dissolved in deionised H20, preparing a stock solution containing 50 g/L 

(nom .) . No signs of toxicity towards the tested strains could be observed : the background 

lawn was visible, the number of revertant colonies was not significantly reduced . 

8 .2 .3 Mutagenicity 

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed . No concentration-related increase over the tested 

range was found. 

Therefore the test item is stated as not mutagenic under the test conditions. 

8 .2 .4 Survey of the findings 

The mean revertant values of the four replicates are presented in the following table. 

Table 8.2-a Mean Revertants Second Experiment 

Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 ~ +S9 -S9 +S9 

H 0 Mean 122 175 12 10 116 124 163 238 10 11 2 sd 35 14 3 6 9 14 35~ 29 4 3 

DMSO Mean 137 154 16 10 

_ 

150 148 192 208 13 12 

sd 23 24 6 2 46 37 45 63 1 4 

Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Pos .Contr. sd 0 0 0 0 0 0 0 0 0 0 

f(l) 7.31 6.50 62.56 100.1 8 .63 6.76 5 .21 4.81 100.1 83.42 

Mean 126 154 10 12 143 147 185 240 14 11 

5031 Nglpl . sd 14 18 2 4 12 50 46 41 3 3 

f(l) 1 .03 0.88 0.83 1 .20 1 .23 1 .19 1 .13 1 .01 1 .40 1 .00 

Mean 102 101 12 10 130 

_ 

153 236 188 9 15 

2516 pg/pl . sd 26 25 5 1 15 52 32 56 2 2 

f(l) 0.84 0.58 1 .00 1 .00 1,12 1 .23 1 .45 0.79 0.90 1 .36 

Mean 140 129 11 10 203 151 228 168 11 13 

1258 Nglpl . sd 57 33 4 5 8 30 13 37 2 0 

f(U ~ 1 .15 1 0.74 1 0.92 1 1 .00 1 .75 1 .22 1 .40 0. 71 1 . 10 1 . 18 

In this table, "1001" represents "> 1000" . 

page 22 of 37 
9 RESULTS 

The test item didn't show mutagenic effects in both experiments . The number of revertant 

colonies was not increased in comparison with the spontaneous revertants (solvent only). 

Cytotoxicity of the test item was not detected. The background lawn was visible and the 

number of revertants was not significantly decreased . 

Therefore it can be stated, that under the test conditions, the test item NHS (Sodium 

hydroxymethanesulfinate) is not mutagenic in the Bacterial Reverse Mutation Test using 

Salmonella typhimurlum, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535. 

14 DISCUSSION 

The test item is considered not mutagenic for the reasons given above . 

Also, the test item didn't show any cytotoxicity towards the bacteria. 

The confirmation tests of the genotype didn't show any irregularities . The control of the 

titre was above the demanded value. The positive controls didn't evoke as many mutations as given by Prof. Ames . But the number of revertant colonies was in the range of the 

historical data of the laboratory (see page 36) and were definitely increased in comparison 

with the negative controls, as well as showing mutagenous potential of the diagnostic 

mutagenes . 

Some of the spontaneous revertants are lower than the values given by Prof. Ames; in 

comparison with the historical data of the LAUS GmbH, they were within the normal range . 

For these reasons, the result of the test is considered valid . 

11 DEVIATIONS 

11 .1 Deviations from the Study Plan 

No deviations from the study plan were observed . 

19 .2 Deviations from the Guideline 

None as known . 

12 RECORDING 

One original of study plan and final report, respectively, all raw data of the study and all 

documents mentioned or referred to in study plan or final report will be kept in the GLP 

Document Archive of the test facility for fifteen years. After that, the sponsor's instructions 

will be applied (shipment of documentation to sponsor) . A retain sample of the test item 

will be kept in the GLP Substance Archive for fifteen years; then, the retain sample will be discarded. 

Number of originals which will be sent to the sponsor: 1 

page 23 of 37 
p. 27 

Final Report of LAUS GmbH 

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13 ANNEX 1 : COPY OF GLP CERTIFICATE 

Gute Laborpnxis I Good Laboratory Practice 

No. : AB07120702G803 

Date: 02. May 2008 

UP-Bescheinigung I Statement of GLP Compliance 

(gem. /according to § 19 Aba. 1 Chemika8engesetz) 

Em* GLP-Inapektlon zur Uberwachung der 

F-arhattung der GLP-Grundeatze qemar6 

Chemikakenpesetz bzw. Richtlime 88I320IEG 

wurde durohgetuhrt in 

Asaessrnant of conformity with GLP according 

lo Chemikakengesetz and Directive ttal340l 

EEC at 

PrUfeinrichtung I Test facility 

LAUS GmbH 

Auf der Schafweide 20 

67489 Kirrevatler 

PrUfung nach Kete(iiorien 1 Areas of Expertise 

(psm I aooordwg CMmVwV-oLP Nr S 3t0ECD qudants) 

1, 3.4, 6, 6, 8 

The abaft maniaHd taq tacky p bRprdad In The nopOnsl 

GLP Caeo4na Pwpramma and is Inspected on a 

npular haut 

aasW on Ors Inpadlon rarwrr M can be eon-IIYmad, trut 

ft reat rapfirY o ate t0 ¢enduW4 tM arorem0alqned 

stunn in wmprlana vrin ft Principlas at GLP 

Varft0tbn of the oomPUance or the tog fatddy wth Re 

Ptit4a<a or the GLP has to be appaed for in UnM t0 atbw 

for a rapow"rq wtapH,y,pn to take OW wRno bog years 

Me, rarnananp" Vhf aOoYf rnnlqrlad "pea., 

ErsPmp rnn Wen, ft WO Whir *0 be taken 001 W OW 

German GLPMqmlorrg Prenrsnrns and ft GLP 

Csrahcate rwaanes wwaea 

Untersc"t, Datum I Signature, Date 

. . . . . .~~. . . . . .,M..~ .o~t .LL~o~- 

INarm und FuMran der rararlwoakMn Perm r narns 

aM Nndbn ar mPSna"' a Parson! 

Dr.-Ing . Karl-Hainz Rother - Prisident - 

l.andesamt fiir Umwefl, Wasserwictschaft und Gewerbeaufsicht 

KaiserFriedrich-Stra(!e 7 

55118 Mainz 

. .. . . . . . .. . . . . . ., . . . . ., . . . . 

Wams WW Adrpsa der GIP- . . . . . . . . . . . . . . . . . . . . . 

Name and aCras of the GLP rdomroriny Auw e~nql r Larnknan8 Illr 

Ltnrnclt . W,t,%crattt'hnll 

unit Gcacrhcaa(sicht 0 

Datum der Inspektion I Date of inspection 

(Tag. Monst Jahr I day .monlh year) 

27 . und 28 . November 2006 

Dfs penanna PNhinrkMvq tfallnoal troh Mm nabonabn 

OLPd7WrwldrutqawMINM und, whd rnpanM6ip aW 

Ernhahunp da GLP.4runafttt utrsrwaahr 

AW der Gtnndtaq. dn tn+PSkfiarwenahN . *Yd hfnmit 

batlript. does In dinar PeWswfeMueq Cat Own 

qaeann~ bn PnrrwrpM udet EeMllunq der GLPGrw~=W LM drpahrhrr warMn bOn»n. 

WhOrdIiMS (Ahptifwg der EInhaNuny der 

GlP-GrunOSllin duroh do PrUhknkMUnq at so 

recM:Mp tu bManusaan. date db FdpskupHnbn 

sWaAens visr Jahn naen darn aaqlnn der o q tnspakhan 

srrirMWan karm 06n dwtM Anlqq whd die 

PqiralnnaMunp naeh I10trvr der Fnsr Bus dam dauMOhen 

GlP-UMmaehunqsprcqrane qMpmmsn und daN Wk 

aesaMinytunp vwrlwA rhre OOKtqhtil 

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14 ANNEX 2: DATA OF THE FIRST EXPERIMENT 

laus .de 

No. : AB07120702G803 

Date: 02. May 2008 

14.1 Determination of Titre 

Criterion : The determination of titre should give a number of at least 109 cells/mL, 

correlating to 100 colonies/plate after dilution. 

Table 14.1-a Titre Values (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 

Re I . 1 -S9 1001 1001 1001 1001 1001 

Re I . 2( -S9) 1001 1001 1001 .1001 1001 

Re ( . 3(+S9) 1001 1001 1001 1001 1001 

Re I . 4( +S9) 1001 1001 1001 1001 1001 

Mean 1001 1001 1001 1001 1001 

sd 0 _ 0 0 0 0 

~ Assessment ~ ok~ ok ok ok ok 

In this table, "> 1000" is represented by "1001". 

14.2 Sterility Control 

Criterion : At the maximum, one colony per plate may grow. 

Table 14.2-a Sterility (colonies per plate) 

H20 DMSO 

Re I . 1 0 0 

Re I . 2 0 0 

Re I . 3 0 0 

Re I . 4 0 0 

~ Assessment ~ ok ok 

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14.3 Spontaneous Revertants 

Criterion : Revertants should lie for TA 97a between 90 and 180, for TA 98 between 30 

and 50, for TA 100 between 120 and 200, for TA 102 between 240 and 320 and for TA 

1535 between 80 and 300 colonies per plate (values by Prof. Ames). 

The determined values were within the normal range of the laboratory. 

14 .3 .1 Water 

Table 14.3-a Spontaneous Revertants H20 (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 1 02 TA 1 535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 

_ 

+S9 -S9 +S9 

Re I . 1 61 90 16 6 203 128 118 211 9 16 

Re I . 2 131 94 7 8 230 190 198 266 8 10 

Re I . 3 163 98 11 8 167 160 

_ 

188 131 10 10 

Re I . 4 167 100 4 14 160 167 181 111 11 10 

Mean 131 96 10 9 190 161 171 180 10 12 

sd ~ 49.1 ~ 4.4 5.2 3.5 32.6 25.6 36.21 71.91 1 .3 1 3.0 

14 .3 .2 DMSO 

Table 14.3-b Spontaneous Revertants DMSO (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Re ( . 1 153 163 15 8 189 194 190 184 17 25 

Re ( .2 150 116 9 10 138 196 177 . 179 15 33 

Re I . 3 78 97 11 10 197 153 178 183 13 10 

Re 1 .4 81 67 12 8 196 183 166 143 10 17 

Mean 116 111 12 9 180 182 178 172 14 21 

sd~ 41 .6 ~ 40.3 ~ 2.5 1,2 28.2 19.8 9.8 196 30 99 

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14.4 Positive Controls 

Without metabolic activation : 

4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 80 Fl,g/plate 

Sodium azide (Na-azide) in deionised water, 6 pg/plate 

With metabolic activation : 

2-Amino anthracene (2-AA) in DIVISO, 3 iag/plate 

Benzo-a-pyrene (BaP) in DMSO, 40 Etg/plate 

Table 14.4-a Diagnostic Mutagenes (colonies per plate) 

Date: 02. May 2008 

Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 

Induction -S9. +S9 -S9 +S9 -So +S9 -S9 +S9 -S9 +S9 

Reference NPD 2-AA NPD BaP Naazide 

2-AA NPD 2-AA Naazide 

2-AA 

Re 1 .1 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Re 1 .2 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Re 1 .3 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Re L.4 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

sd 0 0 0 0 0 0 0 0 0 0 

f(l ) 8.63 9.02 83.42 111 .2 5.27 5.50 5.62 ' 6.82 100,1 47.67 

~ Rev. abs. ~ 885 ~ 890 989 992 811 819 823 829 991 980 ' 

In this table, "> 1000" is represented by "1001". 

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14.5 Test Item NHS (Sodium hydroxymethanesulfinate) 

14.5.1 Mutagenicity Test 

Table 14.5-a Concentration 5047 Nglplate (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Re I . 1 111 114 13 5 169 152 53 165 8 14 

Re I . 2 67 111 5 8 184 151 100 123 16 13 

Re I . 3 182 127 15 10 194 126 104 140 7 15 

Re I .4 117 88 14 6 109 129 70 198 10 20 

Mean 119 110 12 7 164 140 82 157 10 16 

sd 47 16 5 2 38 14 24 33 4 3 

f I 0.91 1 .15 1 .20 0.78 0.86 0.87 0.48 0.87 1 .00 1 .33 

~ Rev. abs. 1 -12 14 2 -2 -26 -21 -89 -23 0 4 

Table 14.5-b Concentration 1514 Ng/plate (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Re L.1 131 144 11 5 182 123 189 196 10 7 

Re ( . 2 110 166 9 13 197 218 194 127 10 7 

Re I . 3 103 124 13 6 221 184 

_ 

111 159 10 8 

Re ( .4 76 115 10 2 129 128 113 61 9 10 

Mean 105 137 11 7 182 163 152 136 10 8 

ad 23 23 2 5 39 46 46 57 1 1 

f( l ) 0.80 1 .43 1 .10 0.78 0.96 1 .01 0.89 0.76 1 .00 0.67 

~ Rev. abs. 1 -26 ~ 41 1 -2 -8 2 -19 -44 0 -4 

Table 14.5-c Concentration 505 pglplate (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Re I . 1 166 110 9 6 180 174 116 109 8 10 

Re I . 2 156 157 7 10 168 129 139 92 9 10 

Re I . 3 94 69 13 7 128 147 111 139 7 10 

Re I .4 129 153 9 7 194 121 135 126 10 9 

Mean 136 122 10 8 168 143 125 117 9 10 

sd 32 41 3 2 28 24 14 20 1 1 

f (1 ) 1 .04 1 .27 1 .00 0.89 0.88 

_ 

0.89 0.73 0,65 0.90 0.83 

Rev. abs. ~ 5 J _ 26 1 0 1 -1 -22 -18 -46 -63 -1 -2 

page 28 of 37 
Table 14 .5-d Concentration 151 Nglplate (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Re 1 .1 157 117 11 11 122 223 106 108 12 10 

Re I . 2 67 112 7 12 95 163 145 131 17 17 

Re I . 3 85 152 11 8 124 180 125 139 5 8 

Re 1 .4 81 110 9 9 195 138 144 150 10 9 

Mean 98 123 10 10 134 176 130 132 11 11 

sd 40 20 2 2 43 36 18 18 5 4 

f( I ) 0.75 1 .28 1 .00 1 .11 0.71 1 .09 0.76 0.73 1 .10 0.92 

Rev. abs. -33 27 0 1 -56 15 -41 -48 1 -1 

Table 14.5-e Concentration 51 pglplate (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1 535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Re I . 1 75 145 12 6 210 138 203 126 10 7 

Re I . 2 133 162 6 6 220 193 186 123 11 8 

Re I . 3 139 110 14 5 191 188 130 156 17 1 0 

Re ( .4 128 140 17 5 149 150 162 190 18 9 

Mean 119 139 12 6 193 167 170 149 14 9 

sd 30 22 5 1 31 27 32 31 4 1 

f(l ) 0.91 1 .45 1 .20 0.67 1 .02 1 .04 0.99 0.83 1 .40 0.75 

Rev. abs. -12 43 2 -3 3 6 -1 -31 4 -3 

page 29 of 37 
Final Report of i_AUS GmbH No. : AB07120702G803 

Auf der Schatweide 20, D-67489 Kimveilet 

Tel . ++49-8321-96299-0, Fax 96299-29, e-mail : infoClaus.de Date: 02. May 2008 

15 ANNEX 3: DATA OF THE SECOND EXPERIMENT 

15.1 Determination of Titre 

Criterion : The determination of titre should give a number of at least 109 cells/mL, 

correlating to 100 colonies/plate after dilution . 

Table 15.1-a Titre Values (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 

Re ( .1 (-S9) 1001 1001 1001 ~1001 1001 

Re ( . 2( -S9) 1001 1001 1001 1001 1001 

Re I . 3( +S9) 1001 1001 1001 1001 1001 

Re l . 4(+S9) 1001 1001 1001 1001 1001 

Mean 1001 1001 1001 1001 1001 

sd 0 0 0 0 0 

Assessment ok ok ok ok ok 

In this table, "> 1000" is represented by "1001 " . 

15.2 Sterility Control 

Criterion: At the maximum, one colony per plate may grow. 

Table 15.2-a Sterility (colonies per plate) 

H20 DMSO 

Re I . 1 0 0 

Re I . 2 0 0 

Re I . 3 0 0 

Re I . 4 0 0 

Assessment ok ok 

page 30 of 37 
Final Report of LAUS GmbH No. : AB07120702G803 

Auf der Schafweide 20, D~67489 ICfnweiler 

Tel. ++49-6321-98299-0, Fax 96299-29, e-mail : infoOlaus.de Date: 02 . May 2008 

15.3 Spontaneous Revertants 

Criterion : Revertants should lie for TA 97a between 90 and 180, for TA 98 between 30 and 50, for TA 100 between 120 and 200, for TA 102 between 240 and 320 and for TA 1535 between 80 and 300 colonies per plate (values by Prof. Ames). 

The determined values were within the normal range of the laboratory. 

15 .3 .1 Water 

Table 15.3-a Spontaneous Revertants HZO (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 ! 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Re I . 1 121 171 15 15 117 104 150 212 11 15 Re I . 2 112 194 10 9 110 131 154 280 -6 8 Re I . 3 85 176 9 15 108 136 135 231 9 13 Re 1 .4 168 160 12 2 128 126 214 229 15 9 

Mean 122 175 12 10 116 124 163 238 10 11 ad 34.6 14.2 2.6 6-2 9.0 14.1 34.8 29.3 3.8 3,3 

15 .3 .2 DMS(J 

Table 15.3-b Spontaneous Revertants DMSO (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 Re I . 1 141 126 16 7 154 170 129 252 13 15 

Re I . 2 104 183 10 11 110 186 206 121 15 9 Re 1 .3 154 144 25 10 122 108 236 258 12 14 Re I . 4 149 161 14 11 213 126 197 200 13 8 Mean 137 154 16 10 150 148 192 208 13 12 

sd 22.6 24.3 6.3 1 .9 46.1 36.6 45.2 63.4 1 .3 3.5 

page 31 of 37 
15.4 Positive Controls 

Without metabolic activation : 

4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 80 gg/plate 

Sodium azide (Na-azide) in deionised water, 6 Etg/plate 

With metabolic activation: 

2-Amino anthracene (2-AA) in DMSO, 3 Ng/plate 

Benzo-a-pyrene (BaP) in DMSO, 40 pg/plate 

Table 15.4-a Diagnostic Mutagenes (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Reference NPD 2-AA NPD BaP Naa zide 

2-AA NPD 2-AA Naazide 

2-AA 

Re 1 .1 1001 1001 1001 1001 

_ 

1001 1001 1001 1001 1001 1001 

Re l .2 1001 1001 1001 1001 1001 1001 1001 1001 1001 , 1001 

Re 1 .3 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Re 1 .4 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

sd 0 0 0 0 0 0 0 ~ 0 0 0 

f I 7.31 6.50 62.56 100.1 8.63 6.76 5.21 4.81 100.1 83.42 

~Rev. abs. ( 864~ 8471 985~ 991 885 853 809 793 991 989 

In this table, "> 1000" is represented by "1001". 

page 32 of 37 
Final Report of LAUS GmbH 

Auf der Schafweide 20, D-87489 Kirrvveiler 

No. : ABU7120702G803 

Tel . ++49-6321-98299-0, Fax 98299-29, e-mail: infa@faus .de I Date: 02. May 2008 

15.5 Test Item NHS (Sodium hydroxymethanesuifinate) 

15.5.1 Mutagenicity Test 

Table 15.5-a Concentration 5031 Nglplate (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 S9 +S9 

Re I . 1 136 148 8 11 141 101 198 229 13 13 

Re I . 2 119 168 8 18 156 115 211 234 17 8 

Re I . 3 138 169 12 8 127 213 215 296 14 13 

Re I . 4 110 131 10 12 149 158 117 199 11 8 

Mean 126 154 10 12 143 147 185 240 14 11 

sd 14 18 2 4 12 50 46 41 3 3 

f(l ) 1 .03 0.88 0.83 1 .20 1 .23 1 .19 1 .13 1 .01 1 .40 1 .00 

LRev. abs. I 4 1 .21 1 .2 1 2 1 - 27 23 22 2 4 0 

Table 15.5-b Concentration 2516 Nglplate (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 

Induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 

Re I . 1 93 82 9 10 124 115 234 180 7 17 

Re 1 .2 112 86 10 11 145 196 282 113 10 16 

Re ( . 3 131 100 10 8 140 199 212 242 11 15 

Re 1 .4 70 136 19 11 

_ 

112 100 217 215 8 12' 

Mean 102 101 12 10 130 153 236 188 9 15 

sd 26 25 5 1 16 52 32 56 2 2 

f l 0.84 0.58 1 .00 1 .00 1 .12 1 .23 1,45 0.79 0.90 1 .36 ~ 

Rev. abs. -20 -74 0 0 14 29 73 -50 -1 4', 

Table 15.5-c Concentration 1258 Nglplate (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 

Induction -S9 +S9 -S9 

_ 

+S9 -S9 +S9 -S9 +S9 -S9 +S9 

Re I . 1 203 134 8 4 210 177 222 197 11 13 

Re I . 2 89 90 9 10 208 113 218 197 9 13 

Re I . 3 175 170 16 15 202 172 225 . 120 12 13 

Re ( .4 94 122 11 12 192 143 247 158 13 13 

Mean 140 129 11 10 203 151 228 168 11 13 

ad 57 33 4 5 8 30 13 37 2 0 

f I 1 .15 0.74 0.92 1 .00 1.75 1 .22 1 .40 0.71 1 .10 1 .18 

~ Rev. abs. ~ 18 1 -46 ~ -1 ~ 0 ~ 87 27 65 -70 1 2 ~ 

page 33 of 37 
Final Report of LAUS GmbH 

Auf der Schafweide 20, D-67489 Kirrweiler 

No. : AB07120702G803 

Tel. ++49-6321-96299-0, Fax 96299-29, e-mail: infoQlaus .de I Date: 02. May 2008 

16 ANNEX 4: DATA OF THE PRE-TEST (CYTOTOXICITY) 

As the pre-test was not performed under GLP-conditions, only the values for the titre and the toxicity control are stated in this report. 

The toxicity of the following concentrations were tested : 

5017 and 1505 lag/plate . 

Per strain, four plates were incubated with the corresponding dose of the test item on 

maximal soft agar. 

The treatments for the confirmation of the genotype, the sterility control and the determi- nation of the titre didn't show any inconsistencies . The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. 

All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation . 

16.1 Experimental Parameters 

Date of treatment January 09th 2008 

Concentrations tested 

Incubation time 

Incubation temperature 

Tester strains 

Method 

16.2 Determination of Titre 

Criterion: 

Table 16.2-a 

5017 / 1505 lag/plate 

48 hours 

37 C 

TA97a, TA98, TA100, TA102, TA1535 

Plate incorporation method 

The determination of titre should give a number of at least 109 ceIIs/mL, 

correlating to 100 colonies/plate after dilution. 

Titre Values (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1536 

Re I . 1 -S9 1001 1001 1001 1001 1001 

Re ( . 2(-S9) 1001 1001 1001 1001 1001 

Re I . 3(+S9) 1001 1001 1001 1001 1001 

Re I . 4(+S9) 1001 1001 1001 '1001 1001 

Mean 1001 1001 1001 1001 1001 

8d 0 0 0 0 0 

Assessment ok ok ok ok ok 

In this table, "> 1000" is represented by "1001" . 

page 34 of 37 
Final Report of LAUS GmbH 

Auf der Schaiweide 20, D-67489 Kirrweiier 

Tel. ++q9-6321-96299-0, Fax 96299-29, e-mail: info@iaus.de 

No. : AB07120702G803 

Date: 02. May 2008 

16.3 Toxicity Control 

The test item is considered non-toxic, if the quotient titreltox is below 2. 

Table 16,3-a 5017 pglplate on maximal-soft-agar (colonies per plate) 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 

Re ( . 1 -S9 1001 1001 1001 1001 1001 

Re I . 2(-S9) 1001 1001 1001 1001 1001 

Re I . 3 +S9 1001 1001 1001 .1001 1001 

Rep] . 4 +S9 1001 1001 1001 1001 1001 

Mean 1001 1001 1001 1001 1001 

sd 0.0 _ _ 0.0 0.0 0.0 0.0 

~ TitrelTox ~ 1 .00 ~ 1 .00 1 .00 1 .00 1 .00 

Table 16.3-b 1505 Nglplate on maximal-soft-agar (colonies per plate) 

Strain TA 97a TA 98 100 TA 102 TA 1535 

Re I . 1 -S9 1001 1001 1001 1001 1001 

Re 1 . 2(-S9) 1001 1001 1001 1001 1001 

Re I . 3(+S9) 1001 1001 1001 1001 1001 

Re I . 4 +S9 1001 1001 1001 1001 1001 

Mean 1001 1001 1001 1001 1001~ 

ad 0.0 0.0 0.0 0.0 0.0 

~ TitrelTox ~ 1 .00 ~ 1 .00 1 .00 1 .00 1 .00 

16.4 Assessment 

On the base of these results, the first experiment was performed with 5000 Ng/plate as 

maximum dose for all strains . 

page 35 of 37 
Final Report of LAUS GmbH 

Auf der schafweide 20, D-67489 Kirnaeiler 

No. : AB07120702Ga03 

Tel. ++49-6321-96299-0, Fax 96299-29, e-mail : info@laus.de I Date: 02. May 2008 

17 ANNEX 5: COMPARISON WITH HISTORICAL DATA 

In the following table, the mean of the spontaneous revertants and positive controls of all 

performed experiments in the year 2007 is stated (Number of experiments : 20) in comparison with the experiments performed within this study. 

Strain TA 97a TA 98 TA 100 TA 102 TA 153 5 

Induction I - S9 +S9 - S9 +S9 - S9 + S9 - S9 +S9 - S9 + S9 

Mean 127 143 24 20 144 151 181 208 13 17 

Min 81 79 11 12 110 131 134 180 6 10 

H20 Max 160 179 53 37 177 180 242 255 26 21 

Ex 1 131 96 10 9 190 161 171 180 10 12 

Ex 2 122 175 12 10 116 124 163 238 10 11 

Mean 134 130 14 15 146 142 186 179 13 13 

Min 94 TA 98 8 6 105 106 120 117 8 5 

DMSO Max 190 182 47 53 204 179 267 277 21 21 

Ex 1 116 111 12 9 180 182 178 172 14 21 

Ex 2 137 154 16 10 150 148 192 208 13 12 

Mean 1001 971 1001 717 1001 1001 1001 1001 1001 908 

Min 1001 467 1001 150 1001 1001 1001 1001 1001 139 

Pos. Contr, Max 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Ex 1 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

Ex 2 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001 

page 36 of 37 
Final Report of LAUS GmbH 

Auf der Schafweide 20. D-67489 Kirrweiier 

Tel. ++49-6321-96299-0 . Fax 96299-29, e-mail : Info 

18 ANNEX 6: GENOTYPE CONFIRMATION 

Date of Performance: January 09th 2008 

18.1 Histidine Requirement 

iaus.de 

No. : AB07120702G803 

Date: 02. May 2008 

Criterion : On the biotin plates, no growth should occur, whereas on the histidine- biotin plates, the strains should grow well. 

Found : Growth on histidine-biotin plates, no growth on biotin plates. 

Assessment: okay 

18.2 Ampicillin-(Tetracyclinee)-Resist . (pKM 101, pAQ1) 

Criterion : After incubation for 12 hours, good growth should be observed on the am- picillin plates for all strains except TA1535. On the ampicillin-tetracycline 

plates, only TA 102 should grow. 

Found: TA97a, TA 98, TA 100, TA 102 grow in the presence of ampicillin, only TA102 with ampicillin and tetracyclinee, TA 1535 doesn't grow anywhere. Assessment: okay 

18.3 UV-Sensitivity (uvrB) 

Criterion : On the irradiated side of the plates, only TA 102 should grow, whereas on the side, which was protected from light, all strains should grow. Found : all strains grow on the non-irradiated half, only TA102 on the irradiated 

one . Assessment : okay. 

18.4 Crystal Violet Sensitivity (deep roughirfa) 

Criterion : The zone of inhibition has a diameter of at least 14 mm. 

Found : 

Table 18.4-a Inhibition in mm 

Strain TA 97a TA 98 TA 100 TA 102 TA 1535 Re l . 1 15 14 15 16 15 Re I . 2 15 14 16 16 15 Mean 15 14 15.5 16 15 Assessment: okay. 

page 37 of 37 
Endpoint study record: Genetic toxicity in vitro.001 file :///C:/Dokumenke >,nd EinstellungenPil/Desktop/index.htrnl 

Printing Date 2011-03-07 13 :11:33 CET 

Restriction of specific regulatory purposes 

EU: BPD, EU: PPP, EU: REACH, CA: CEPA, CA: PCPA, JP : CSCL, OECD: HPVC, US : EPA HPVC. US: FIFRA, US: TSCA, other. 

Confidentiality 

CBI, IP, no PA 

Owner Sodium hydroxymettmnesulphinate / sodium hydroxymelhanesulphinate / sodium hydroxymeMianesulfinate / 149-44-0 / 

Briiggemannehemical, L. Bruggemann KG / Heilbronn / Germany 

Legal entity Biiiggemannehemical L. Briiggemann KG / Heilbronn / Gemiany 

owner 
Endpoint study record : Genetic toxicity in vitro.001 fi1e :///C:/Dokumente ind Einstellungen&/tesktop/indexhtml 

Endpoint study record: Genetic toxicitv in vitro.001 

UUID IUC5-9bbede48-512a-4ace-a523-80320135dedd 

Dossier UUID 0 

Author UL / BruggemannChemical L. Briiggemann KG / Heilbronn / Germany 

Date 2011-03-07 13 :10 :57 CET 

Remarks 

Administrative Data 

EU: REACH 

Purpose flag key study, robust study summary; used for classification ; used for MSDS 

Study rosuRtype experimental result Study period January 2008 - May 2008 

Reliability 1 (reliable without restriction) 

Rationale for GLP guideline study. Planning and conduct of the study were based on the OECD guideline "Genetic Tobcology. Salmonella 

reliability incl . typNmurium, Reverse Mutation Assay", OECD No . 471 OECD Guidelines forttie Testing of Chemicals Bacterial Reverse 

deficiencies Mutation Test' Part 471, adopted July 21 st 1997 EU-Guideline 67/548 EWG, last changed by 29th amendment Annex V, 

Method B.13/14 "Mulagenicity- Salmonella typhimurium, reverse mutation assay, adopted May 191h, 2000 

Data source 

Reference 

Data access 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report no . Ownercompany Company 

study no . 

Report 

date 

study Paulus, 2008 Determination ofthe LAUS AB07120702G803 BruggemamChemical, 2008_05_02_01 2008-05-20 

report Jbrg mulagertic potential of GmbH; L.Bruggemann KG 

NHS (Sodium Kimveiler, 

hydroxymethanesuffinate) Germany 

with the Bacterial 

Reverse Mutation Test 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Type of genotoxicity 

gene mutation 

Type of study 

bactedal reverse mutation assay (e.g . Ames best) 

Test guideline 

Qualifier Guideline Deviations 

according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) (OECD 471) no 

I according to EU Method 8.1 3/14 (Mulagenicity- Rev erse Mutati on Test Using Bacteria) no 

GLP compliance 

yes (incl . certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Confidential details on test material 

Lot no . :07041205 

Method 

Target gene 

his-of Salmonella typhimirium LT2 

Strains: TA 1535, TA 97a, TA 98, TA 100 and TA 102 

Species/strain 

Specieslstraln S. typhimurium TA 98 

Additional other. hisD3052, uvr8, pKM 101, rfa 

strain 

characteristics 
Enapoint study record : Genetic toxicity in vitro.001 file :///C:/Dokwnente wid Einstellunw~genul/Desktop/indexhtml 

Metabolic with and without 

activatbn 

Metabolic Rat liver S9 metabolic activation 

activation 

system 

Specleslstrain S. typhimurium, other. TA97a 

Additional other. hisD6610, uvrB, pKM 101, rfa 

strain 

characteristics 

Metabolic with and without 

activation 

Metabolic Rat liver S9 metabolic activation 

activation 

system 

Specles/straln S. typhimurium TA 100 

Additional other. hisG46, uorB, pKM 101, rfa 

strain 

characteristics 

Metabolic with and without 

activation 

Metabolic Rat lirer S9 metabolic activation 

acfhratbn 

system 

Specles/strain S. typhimurium TA 102 

Additional other. hisG428, pKM 101, rfa 

strain 

characteristics 

Metabolic with and without 

activation 

Metabolic Rat liwr S9 metabolic activation 

activation 

system 

Specleslstrain S. typhimurium TA 1535 

Additional other. HisG46 

strain 

characteristics 

Metabolic with and without 

activation 

Metabolic Rat liver S9 metabolic activation 

activation 

system 

Test concentrations 

Experiment I (plats incorporation mettiod): conc : 5047 / 1514 / 505 / 151 / 51 Ng/plats 

Experiment II (pre-incubation method): conc. : 5031 /2516 / 1258 Ng/plate 

vehicle 

-vehide(sysolvenE(s) used : [water] 

- Justification for choice of sohenf/vehicle : test item was completely soluble in water, and water does not ham any efects on the viability of the bacteria 

or the number of spontaneous reoertanFs . 

Controls 

Solvent/ yes (water) 

vehicle 

controls 

Positive yes (TA 97a, TA 98 and TA 102) 

controls 

Positive other. 4Nitno-1,2-pherrylene diamine (80 Ng/plate) 

control 

substance 

Remarks without metabolic activation system 

Solvent/ yes (water) 

vehicle 

controls 

Positive yes (rA 100 and TA 1535) 

controls 

Positive sodium aade (8 Ng/plate) 

control 

substance 
Endpoint study record: Genetic toxicity in vitro.001 file :///C:/Dokuniente und Einstellungenul/Deslctop/index.html 

Rernarks without rnstabolicadivation system 

Solvent l yes (water) 

vehicle 

controls 

Positive yes (TA 98) 

controls 

Positive benao(a)pyrore (40 Nglplate) 

control 

substance 

Remarks with metabolic activation system 

Negative yes 

controls 

Solvent/ nodafa 

vehicle 

controls 

True yes 

negative 

controls 

Positive yes (TA 97a, TA 100, TA 102 and TA 1535) 

controls 

Positive other. 2-AminoardFuacene (3 Nglplate) 

control 

substance 

Remarks with metabolic activation system 

Details on test system and conditions 

METHOD OF APPLICATION: in agar (plate incorporation) 

- Preincubation period : 12 hours. 

PLATE INCORPORATION METHOD (First Experiment): 

- Per strain and dose, four plates with and four plates without S9 mix were used. 

10 mL of the test solution of to appropriate concentration were membrane flllratsd into sterile vessels. 

Top agar basis was melted in a microwave oven, after melting, 10 mL of histidinebiotin-solution 0.5 mMol per 100 mL basis was added and the bottle 

was placed into water bath at 45 °C . 0.1 mL of the appropriate solution of the test item were given into a sterile tube. After mixing with 0.1 mL 

overnight culture of the respective strain and 0.5 mL phosphate buffer (only for treatmerb without S9) or 0.5 mL S9 mix, 2 ml. Top-Agar were added. The mixture was gently eortexed, then poured on a minimal glucose plate and distributed erenly,using a Drigalski Spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C . 

PRE-INCUBATION METHOD (second experiment): 

- Per stain and dose, four plates with and four plates without S9 mixwere used. 

10 mL of the test solution of the appropriate concentration were membrane filirated into sterile vessels. Top agar basis was melEed in a microwave 

oven, after melting, 10 ml. of Itistidinebiotin- solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the water bath at 45 °C . 

0.1 mL of the appropriate solution of the test item were given into a sterile tube . After mixing with 0.1 ml. overnight culture of the respective strain, 0.5 

mL phosphate buffer (only for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 2 mL top agarwere added. The mixture was oortexed gently, then poured on a minimal glucose plate and distributed eoerNy, using a Drigalski spatula. The plates were dosed, covered with brown paperand leftto harden for a few minutes, then inverted and placed in the dark incubator at 37 °C . 

NUMBER OF REPLICATIONS : 

Four replicates, withMthout S9, for each solvent which was used in the test 

Evaluatlon criteria 

A test item is considered to have muFagenic potential, if a significant, reproducible increase of revertant colonies per plate (ncrease factor 2) in at least 

one strain can be observed . A concentration-related increase over the range tested can also be taken as a sign of mutagenic actioity . 

Statistics 

STATISTICAL METHDODS USED: 

The colonies were counted visually, the numbers were recorded . A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations as well as the increase factor of revsrtant induction . 

Results and discussions 

Test results 

Speciieslstrain other: TA 97a, TA 98, TA 100, TA 102 and TA 1535 

Metabolic with 

activation 

Test systam all strains/call types tested (First Experiment) 

Genotoxicity negative 

Cytotoxk:Uy no 

Vehicle yes 

controls valid 

Negative yes 

controls valid 

Positive yes 

controls valid 
Endpoint A r% study record : Genetic toxicity in vitro.001 file :///C:/Dokumente und Einstellungenul/Ifesktop/index .html 

Species/strain other. TA 97a, TA 98, TA 100, TA 102 and TA 1535 

Metabolic without 

activation 

Test system all strains/csl types tested (First Experiment) 

Genotoxicity negative 

Cytotoxicily no 

Vehicle yes 

controls valid 

Negative 

controls valid 

yes 

Positive yes 

controls valid 

Specieslstrafn other. TA 97a, TA 98, TA 100, TA 102 and TA 1535 

Metabolic with 

activation 

Test system all strains/cell types tested (Second Experiment) 

Genotoxicity negative 

Cytotoxiclly no 

Vehicle yes 

controls valid 

Negative 

controls valid 

Positive 

controls valid 

yes 

yes 

Specieslstraln other. TA 97a, TA 98, TA 100, TA 102 and TA 1535 

Metabolic without 

activation 

Test system all strainslcell types tested (Second Experiment) 

Genotoxicity negative 

Cytotoxicity no 

Vehicle yes 

controls valid 

Negative yes 

controls valid 

Positive yes 

controls valid 

Additional informatlon on resnlts 

OBSERVATION 

FIRST EXPERIMENT: 

" The treatments for the confirmation of the genotype, the sterility control and the determination of the titer did not show any inconsistencies. " All positive controls (diagnostic mulagens) showed mulagenic effects with and without metabolic activation. " No signs of toxicity towards the tested strains could be observed : 

" The background lawn was visible; the number of revertant colonies was not significantly reduced. 

" No significant increase of the number of reverlant colonies in the treatments with and without metabolic activation could be observed . " No concenh-ation-related increase over the tested range was found. 

SECOND EXPERIMENT: 

" The treatments forthe confirmation of the genotype, the stsrility control and the determination of the titer did not show arry inconsistencies . " All positive controls showed mulagenic effects with and without metabolic activation. 

" No signs of toxicity towards the tested strains could be observed: 

" The background lawn was visible; the number of reoertant colonies was not significantly reduced. 

" No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed . " No concentration-related increase over the tested range was found. 

COMPARISON WITH HISTORICAL CONTROL DATA: 

The determined values for the spontaneous revertards of the negative controls were in the normal range of the test laboratory historical data of the laboratory (in the first and in the second experiment). 

Applicant's summary and conclusion 

Interpretation of results 

negative with metabolic activation 

negative without metabolic activation 

Conclusions 

The test item is considered to ben non-mufagenic in the bacterial reverse mutation test 

Executive sunriffiary 

First experiment 
Er~dpoint study record : Genetic toxicity in vitro.001 file :///C:/Dolamnente und EinstellungeNul/4D6eslctop/index.html 

Five concentrations of the test item, dissolved in deionised water (ranging from 5047 to 51 ug/plate) were used . Five 

genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method. 

None of the concentrations caused an increase in the number of rewertant colonies in the tested strains. 

The test item didn't show any mutagenic effects in the first experiment . The test item didn't show any cytotobcity towards the bacteria . 

The sterility control and the determination of the titer did not show any inconsistencies . The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. 

Second exoeriment 

To verify the results of the first experiment, a second experiment was performed, using three concentrations of the test item (ranging from 5031 to 1258 Ng/plate) and a modification in a study performance (pre-incubation method). 

The test item didn't show any mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed . 

The sterility control and the determination of the titer did not show any inconsistencies . The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive showed mutagenic effects with and without metabolic activation . 

Under the conditions of the test, the test item did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535 . Therefore, no concentration-effect relationship could be determined . 

The test item is considered as "not mutagenic" under the conditions of the test .